Synapse specificity of calcium release probed by chemical two-photon uncaging of inositol 1,4,5-trisphosphate

Biological messengers can be "caged" by adding a single photosensitive group that can be photolyzed by a light flash to achieve spatially and temporally precise biochemical control. Here we report that photolysis of a double-caged form of the second messenger inositol 1,4,5-trisphosphate (IP₃) triggers focal calcium release in Purkinje cell somata, dendrites, and spines as measured by two-photon microscopy. In calbindin knock-out Purkinje cells, peak calcium increased with flash energy with higher cooperativity for double-caged IP₃ than for conventional single-caged IP₃, consistent with a chemical two-photon effect. Spine photolysis of double-caged IP₃ led to local calcium release. Uncaging of glycerophosphoryl-myo-inositol 4,5-bisphosphate (gPIP₂), a poorly metabolizable IP₃ analog, led to less well localized release. Thus, IP₃ breakdown is necessary for spine-specificity. IP₃- and gPIP₂-evoked signals declined from peak with similar, slow time courses, indicating that release lasts hundreds of milliseconds and is terminated not by IP₃ degradation but by intrinsic receptor dynamics. Based on measurements of spine-dendrite coupling, IP₃-evoked calcium signals are expected to be at least 2.4-fold larger in their spine of origin than in nearby spines, allowing IP₃ to act as a synapse-specific second messenger. Unexpectedly, single-caged IP₃ led to less release in somata and was ineffective in dendrites and spines. Calcium release using caged gPIP₂ was inhibited by the addition of single-caged IP₃, suggesting that single-caged IP ₃ is an antagonist of calcium release. Caging at multiple sites may be an effective general approach to reducing residual receptor interaction.